Fujisawa K, Fujita A, Ishizaki T, Saito Y, Narumiya S
Department of Pharmacology, Kyoto University Faculty of Medicine, Sakyo-ku, Kyoto 606, Japan.
J Biol Chem. 1996 Sep 20;271(38):23022-8. doi: 10.1074/jbc.271.38.23022.
A protein serine/threonine kinase, p160(ROCK), has been identified as a putative Rho target protein that is activated when bound to the GTP-bound form of the small GTPase Rho (Ishizaki, T., Maekawa, M., Fujisawa, K., Okawa, K., Iwamatu, A., Fujita, A., Watanabe, N. Saito, Y., Kakizuka, A., Morii, N., and Narumiya, S. (1996) EMBO J. 15, 1885-1893). p160(ROCK) has a serine/threonine kinase domain in its NH2-terminal region, followed by an approximately 600-amino acid-long alpha-helix, a cysteine-rich zinc finger-like motif, and a pleckstrin homology region in the COOH terminus. To identify the Rho binding domain of this protein, we divided p160 into five fragments, expressed each as a His-tagged recombinant protein, and performed a ligand overlay assay using [35S]guanosine-5'-3-O-(thio)triphosphate (GTPgammaS)-bound glutathione S-transferase-RhoA. Specific GTPgammaS-Rho binding was observed only in the fragment M2, which covered most of the carboxyl half of the alpha-helix between amino acids 727 and 1021. This fragment was further subdivided into several fragments, and the ligand overlay assay as well as the yeast two hybrid system was carried out to identify the Rho-binding region. These studies localized the minimum Rho binding region to amino acids 934-1015. To identify critical amino acids for Rho binding, we analyzed the Rho binding activity of the subfragment with various point mutations. This analysis revealed that K934M, L941A, and E1008A mutations significantly weakened Rho binding and an I1009A mutation abolished Rho binding. The amino acid sequence in this region had no significant homology with Rho effector motif class 1, which is shared by putative Rho targets, PKN, rhophilin, and rhotekin, (Reid, T., Furuyashiki, T., Ishizaki, T., Watanabe, G., Watanabe, N., Fujisawa, K., Morii, N., Madaule, P., and Narumiya, S. (1996) J. Biol. Chem. 271, 13556-13560) and may define a distinct class of Rho effector motif.
一种蛋白丝氨酸/苏氨酸激酶，p160(ROCK)，已被鉴定为一种假定的Rho靶蛋白，当它与小GTP酶Rho的GTP结合形式结合时被激活（石崎彻、前川真、藤泽和子、小川健、岩松晃、藤田明、渡边直、斋藤洋、柿冢明、森井直和鸣宫茂，(1996)《欧洲分子生物学组织杂志》15, 1885 - 1893）。p160(ROCK)在其NH2末端区域有一个丝氨酸/苏氨酸激酶结构域，接着是一个约600个氨基酸长的α螺旋、一个富含半胱氨酸的锌指样基序以及COOH末端的一个普列克底物蛋白同源区域。为了鉴定该蛋白的Rho结合结构域，我们将p160分成五个片段，每个片段都作为带His标签的重组蛋白进行表达，并使用[35S]鸟苷 - 5'-3 - O - (硫代)三磷酸(GTPγS)结合的谷胱甘肽S - 转移酶 - RhoA进行配体覆盖分析。仅在片段M2中观察到特异性的GTPγS - Rho结合，该片段覆盖了氨基酸727至1021之间α螺旋羧基端的大部分区域。该片段进一步细分为几个片段，并进行配体覆盖分析以及酵母双杂交系统以鉴定Rho结合区域。这些研究将最小的Rho结合区域定位到氨基酸934 - 1015。为了鉴定Rho结合的关键氨基酸，我们分析了具有各种点突变的亚片段的Rho结合活性。该分析表明，K934M、L941A和E1008A突变显著削弱了Rho结合，而I1009A突变则消除了Rho结合。该区域的氨基酸序列与假定的Rho靶蛋白PKN、亲Rho蛋白和Rho结合蛋白所共有的1类Rho效应基序没有显著同源性（里德、古矢敷树、石崎彻、渡边刚、渡边直、藤泽和子、森井直、马达勒、鸣宫茂，(1996)《生物化学杂志》271, 13556 - 13560），可能定义了一种独特的Rho效应基序类别。
